PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression

Background: The inward rectifier potassium current I_K1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased I_K1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC₅₀ = 14 nM with inside-out patch clamp methodology) and specific I_K1 inhibitor that interacts with the cytoplasmic pore region of the K_IR2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) K_IR2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N K_IR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations.

Methods: Molecular modelling was performed with the human K_IR2.1 closed state homology model using FlexX. WT and mutant K_IR2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. K_IR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively.

Results: PA-6 docking in the V93I/D172N double mutant homology model of K_IR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC₅₀ = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC₅₀ = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward I_K1 at -50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased K_IR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular K_IR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM).

Conclusions: 1) KCNJ2 gain-of-function mutations V93I and D172N in the K_IR2.1 ion channel do not impair PA-6 mediated inhibition of I_K1, 2) PA-6 elevates K_IR2.1 protein expression and induces intracellular K_IR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.